NK cell preparation
Haploidentical related donors were chosen, with preference given to killer immunoglobulin-like receptor (KIR) ligand-incompatible donors. They underwent lymphapheresis for 3–5 hours on the day prior to cell infusion (day–1). Peripheral blood mononuclear cells (PBMC) were collected using a Fenwal CS-3000 Plus blood cell separator (number 4R4538) with a granulocyte separation chamber and small volume collection chamber. Up to 2×1010 PBMC were incubated with magnetic-activated cell sorting (MACS) colloidal super- paramagnetic CD3 Micro-Beads, which consisted of monoclonal mouse anti-human CD3 antibodies conjugated to microspheres. T cells were depleted from the lymphopheresis product using the Miltenyi Biotec CliniMACS cell selection device under good manufacturing practice (GMP) conditions. All cell products contained more than 70% viable cells for infusion.
Cells were stained with the following monoclonal antibodies (MAb): peridinin chlorophyll A protein (PerCP)-conjugated SK7 (anti-CD3), allophycocyanin (APC)-conjugated NCAM16.2 (anti- CD56), APC- conjugated SK3 (anti-CD4), phycoerythrin (PE)- conjugated 2A3 (anti-CD25), APC-conjugated M-A251 (anti-CD25), PE-conjugated h-IL7R–M21 (anti-CD127), fluorescein isothiocyanate (FITC)-conjugated eBioRDR5 (anti-CD127) and Alexa Fluor 488-conjugated 259D (anti-Foxp3) as indicated. IL-15 and IL-7 concentrations were determined on frozen serum and plasma, respectively.
NK cells were administered by intravenous infusion 2 days after the last dose of fludarabine. On the evening of the cell infusion, patients began subcutaneous injections of IL-2 (10 MU) given three times weekly for a total of six doses, based on regimens known to expand autologous and allogeneic NK cells in vivo. Seven patients also received 200 cGy TBI on day 1. TBI was delivered with a linear accelerator using 6, 18 or 25 MV photons in a single fraction on day–1, with right and left lateral fields at a dose rate between 10 and 19 cGy/min prescribed to the midplane of the patient at the level of the umbilicus.
Toxicity and response
Expected low-grade (1 and 2) toxicities were observed in most patients. Eleven patients developed grade 3 toxicity, and none of the expected side-effects reached grade 4. Despite the known constitutional symptomsassociated with IL-2, injections were tolerable, with 93% (111/120) of the planned IL-2 doses administered. Sixteen patients received all six planned doses. Fewer doses were given to one patient with high fever (five doses), one with hypoxia (five doses) and one with autoimmune hemolysis (four doses).
There were 10 unanticipated severe adverse events (SAE) (four grade 3, five grade 4 and one grade 5). Eight of these occurred following the addition of TBI. Two of the SAEs (decrease in cardiac ejection fraction and abdominal pain), although unexpected, resolved with no permanent sequelae. The grade 5 toxicity was a death attributed to tumor lysis syndrome (TLS) in a 51-year-old woman with significant ovarian cancer tumor burden (maximal size liver metastasis 6.8×4.8 cm).
Five patients had neutropenia beyond day +28, four of whom received TBI. There were no significant differences between the breast and ovarian cohorts with respect to time to neutrophil or platelet recovery or red blood cell (RBC) transfusion independence. However, patients who received TBI had significantly longer median times to neutrophil recovery [TBI 32 days: no TBI 15 days). Similarly, the median times to transfusion independence were longer after TBI: platelets (TBI 51 days; no TBI 18 days) and RBC (TBI 43 days; no TBI 15 days).
Re-evaluation of disease by computed tomography (CT) scan was performed 4–6 weeks following NK infusion and every 3 months thereafter. There were 4 patients with partial response (PR) (all ovarian), 12 with SD (8 ovarian and 4 breast) and 3 with PD (1 ovarian and 2 breast) at a median of 36 days (range 31– 109) following NK cell infusion. The median time to progression was 2 months (range 1–6).
Adoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.
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Scientific article publishing date 9/20/2010
Immucura identifier BSC21_276EN